Accurate analysis of nucleic acids (single- and double-stranded DNA or RNA) is of central importance for understanding life processes in cells and, in particular, for identifying the information contained in the DNA.
Methods have already been developed for analyzing nucleic acid sequences. The classical method for sequencing (Proc. Natl. Acad. Sci. USA 74 (1977) 5463) nucleic acids, especially DNA, is slow, susceptible to error and very difficult to automate. This is why there has been development recently of some methods which employ fluorescence labeling of DNA (J. Biochem. Biophys. Meth. 13 (1986) 315, Nature 321 (1986) 674, Science 238 (1987) 336). However, none of these methods makes use of specific interactions between dye and nucleic acid in order to characterize the DNA. Hence all the known methods require either several dyes or several lanes in the separation medium.
The use of thionucleotides for DNA sequencing by limited exonuclease digestion is described in WO 86/07612. However, this method is not very suitable for automation.
Furthermore, thionucleotides are employed in investigations of cells because of the limited digestability by exonucleases (Nucleic Acids Res. 18 (1990) 829). Reports of the use of aminonucleotides as terminators in DNA sequence analysis have mentioned only radioactive detection, with an indication of the possibility of coupling other detector molecules to the free amino group (Nucleic Acids Res. 12 (1984) 1671).
Moreover, several methods have been developed for visualizing DNA in in situ hybridization (Proc. Natl. Acad. Sci. USA 78 (1981) 6633, Exptl. Cell. Res. 153 (1984) 61, Histochem. 85 (1986) 1). However, these methods are very time-consuming or, owing to unavoidable deficiencies, give results which are unreliable in some cases.
DNA in which thionucleotides are incorporated can be detected in gels by coupling suitable fluorescent dyes (Biochem. 28 (1989) 261). However, the dye-labeled single-strand DNA is unstable under the conditions of electrophoresis and can therefore not be isolated pure as a fluorescent probe for the hybridization.
A method for the fluorescence labeling of nucleic acids by enzymatic transfer of a dye-labeled thionucleotide has also been described (Nucleic Acids Res. 12 (1984) 1791). However, a maximum of only one dye molecule per DNA strand can be coupled to the DNA by this method.